Immunoassay method and apparatus

ABSTRACT

An improved immunoassay sample determination process for determining the presence of a component of an antigen-antibody reaction in a sample is disclosed which substantially eliminates non-specific interactions between the sample and the reaction vessel wall surfaces during the antigen-antibody reaction, to thereby greatly increase the accuracy of the process. In practice, a site-deactivating medium such as an animal- or vegetable-derived total biological fluid or extract (e.g., plasma or gelatin) is covalently bound to the vessel wall surfaces for deactivation purposes. In preferred forms the process is solid-state, wherein one of the components of the antigen-antibody reaction is coupled covalently to the coating medium, and a reaction mixture fraction including the sample being determined and the other component which has been labeled, for instance by means of a colorimetrically active enzyme.

This is a continuation of application Ser. No. 06/040,641, filed on May21, 1979, U.S. Pat. No. 4,267,270.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly concerned with improved immunoassayprocesses wherein use is made of a site-deactivating medium for reducingor substantially eliminating non-specific interactions between a samplebeing tested and the surfaces of the reaction vessel. More particularly,it is concerned with an improved method of this type wherein the vesselwall surfaces in contact with the sample and antigen-antibody componentsduring the reaction are coated and covalently bound with a medium suchas a total biological fluid or extract for site-deactivation purposes.

2. Description of the Prior Art

A number of immunoassay techniques have been proposed in the past fordetermining, either qualitatively or quantitatively, the presence of acomponent of an antigen-antibody reaction in a given sample. Forexample, immunodiffusion and immuno-electrophoresis, complementfixation, passive hemagglutination and radio-immunoassay procedures havebeen developed. Furthermore, U.S. Pat. No. 3,654,090 describes aimmunochemical assay process wherein use is of a component of anantigen-antibody reaction in insolubilized form, whereas the othercomponent is covalently linked to an enzyme. The insolubilized and freeenzyme-labeled components are added to a sample to be determined, andthe antigen-antibody reaction is allowed to proceed to completion. Ifthe sample contains one of the components of the antigen-antibodyreaction, this component competes with the corresponding added componentfor reaction with the other component of the reaction. When theantigen-antibody reaction is complete, the insolubilized and freefractions are separated, and the activity of the labeled component isdetermined by an appropriate measure of one of the separated fractions.

Solid-state techniques analogous to that outlined above are inwidespread use. In such processes, one of the components of anantigen-antibody reaction is adsorbed onto an insoluble carrier orsurface in a reaction vessel; for example, the component can simply beadsorbed directly to the wall surfaces of a synthetic resin vessel. Areaction mixture fraction is then added to the vessel which wouldinclude the sample being determined and the other component of theantigen-antibody reaction which has been labeled. Such labeling can beaccomplished by a number of means, for example using a radioisotope orcovalently linking an enzyme to the free component. The antigen-antibodyreaction is then allowed to proceed, wherein one of the followingpossibilities can occur: (1) If the sample being determined is free ofthe labeled component, then all of the latter will react with theinsolubilized fraction, and the remaining reaction mixture fraction willbe free of antigen; or (2) If the sample contains a quantity of thecomponent corresponding to the labeled component, a competition resultsbetween the labeled component and that in the sample. In this case,because of such competition, an amount of the labeled component willremain in the reaction mixture fraction. In either event, the remainderof the reaction mixture fraction and the insolubilized fractions areseparated, and the label activity is measured on one of these fractions.Determination of activity of course depends on the nature of the label;in the case of a radioisotope, an isotope counter is employed, whereasif an enzyme label is employed, enzyme activity may be determinedcolorimetrically.

While the above described solid-state assay procedures are known, apersistent problem which has detracted from the usefulness thereofinvolves non-specific interaction which can occur between components ofthe sample and the reaction vessel wall surfaces and/or other adsorptionsurfaces present during the antigen-antibody reaction. As can beappreciated, such non-specific interaction can materially detract fromthe accuracy of the solid-state technique, particularly if quantitativedeterminations are desired.

To give but one example of this problem, many insurance companies todayrequire a urine sample from applicants for their policies, and suchsamples are often checked for the presence of thiazides therein. If suchthiazides are present, a good indication is given as to whether theapplicant is taking certain types of medications. In any event, use ofthe above-described solid-state technique in connection with thiazidedeterminations presents significant problems relating to non-specificinteractions, which is enhanced because of varying quantities of ureapresent in the urine samples. In fact, such interactions can be sosignificant as to lead to totally erroneous qualitative results, i.e., aurine sample free of thiazide tests as a positive, or athiazide-containing sample tests as a negative.

There is therefore a decided need in the art for an improvedimmunochemical assay process which overcomes the problems associatedwith non-specific interactions, particularly in the case of solid-stateimmunoassay techniques.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is concerned with a process for the determinationof the presence of a component of an antigen-antibody reaction in asample. In its particular aspects, the invention contemplates the use ofa site-deactivating medium in the process for the purpose of eliminatingor at least substantially minimizing non-specific interactions betweenthe sample being tested and the defining walls of the reaction vesseland/or other possible adsorption surfaces therein.

In accordance with the invention, respective, calculated quantities ofthe antigen component and the antibody component of the reaction areprovided, with one of the components being labeled.

It is to be understood that analogues of tested for compounds may beused in this context, as long as the analogue and the compound beingtested for have comparable biological activities. To give but oneexample, in a test for the presence of a thiazide antigen in a urinesample, a thiazide analogue antigen and corresponding antibody can beused. As used herein, the step of providing antigen and/or antibodycomponents of a given reaction shall be taken to include provision ofsuch biologically similar analogues.

Either the antigen or antibody component can be labeled, but preferablythe antibody is labeled. Likewise, a variety of labels can be employed,such as a radioactive isotope or a color-active enzyme; in preferredforms, a color-active enzyme is preferred.

In addition, a reaction vessel is provided which is defined by wallsurfaces which will contact the reactants and sample employed in thetest procedure. A multiple-well microtiter plate is advantageously usedas the reaction vessel, especially in conjunction with screening testsfor a large number of samples. In this case, the defining walls of therespective wells can present interference problems, as will be explainedin detail hereinafter.

In the process, the defining wall surfaces of the plate wells are coatedby covalently bonding thereto a site-deactivating medium which serves,during the antigen-antibody reaction, to minimize non-specificinteractions between these surfaces and the sample being tested. Thesite-deactivating medium is selected from the group consisting ofanimal-derived total biological fluids, and extracts thereof,vegetable-derived total biological fluids, and extracts thereof, andmixtures of any of the foregoing. Preferably, the medium should be watersoluble so as to facilitate the covalent bonding thereof to the surfaceor surfaces.

One of the provided antigen or antibody components is insolubilized,most preferably by covalently bonding the selected component directly tothe site-deactivating medium. Preferably, the antigen component of thereaction is insolubilized.

The next step involves adding a reaction mixture fraction to the vesselwhich includes the sample being tested and the noninsolubilizedcomponent which has been provided. In the preferred process, thenoninsolubilized component would be the antibody, and this antibodywould in turn be the component which had been labeled.

The antigen-antibody reaction is then allowed to proceed to completionin the vessel. As described above, there are two possibilities inconnection with this reaction. That is to say, if the sample beingtested does not contain the tested for component of the antigen-antibodyreaction, all of the provided antigen and antibody will react. On theother hand, if the sample contains the tested for component, acompetition will result for reaction with the remaining component. Inthe case of the preferred process wherein the antigen is insolubilizedand a labeled, soluble (i.e., noninsolubilized) antibody is placed inthe reaction vessel along with the sample, the following can occur. Ifthe sample contains the antigen being tested for, there will be acompetition between the antigen within the sample and the insolubilizedantigen for reaction with the labeled antibody. If none of the antigenbeing tested for is present, all of the labeled antibody will react withthe insolubilized antigen.

The final steps of the process involve separating whatever remains ofthe reaction mixture fraction from the insolubilized fraction andwhatever has reacted with the latter, followed by determining whetherthe tested for component of the antigen-antibody reaction is present inthe sample by determining the activity of the labeled component in oneof the separated fractions. Again referring to the most preferredprocess, the determination procedure involves contacting acolor-activating material with the insolubilized fraction and whateverhas reacted with the latter. If no tested for antigen is present in thesample, a first color reaction will obtain because of the fact that allof the labeled antibody has reacted with the insolubilized antigen. Ifthe sample contains the tested for antigen, less of the labeled antibodywill have reacted with the insolubilized antigen (by virtue of thecompetition for reaction with the antigen between the labeled antibodyand the antigen in the sample), and therefore a different amount ofcolor reaction will obtain.

The most preferred labeling enzyme is horseradish peroxidase, whereasthe most preferred site-deactivating medium is selected from the groupconsisting of animal-derived plasma, animal-derived sera,vegetable-derived gelatins, and mixtures of the foregoing.

The following example will illustrate in detail a process in accordancewith the invention. It is to be understood however, that the example isfor illustrative purposes only, and nothing therein should be taken as alimitation upon the overall scope of the invention.

EXAMPLE

The present invention is eminently suited for large scale determinationof the presence of thiazides in human urine samples. In a representativeprocedure, multiple-well synthetic resin microtiter plates are employedwhich serve as reaction vessels; and the defining walls of therespective wells also present insolubilizing surfaces used in thedetermination. Moreover, a thiazide analogue is used as the antigen, anda thiazide antibody covalently linked to horseradish peroxidase (HRP)enzyme is used as the labeled antibody.

The HRP-labeled antibody is made as follows. A quantity of the antigenanalogue,3-(β-carboxyethyl)-6-chloro-7-sulfamoyl-1,2,4-benzothiadiazine-1,1-dioxide(see U.S. Pat. No. 3,287,360) is reacted with bovine serum albumin(BSA), using 64.5 mg. of the thiazide compound and 200 mg. of BSA in 8ml. distilled water. 40 mg. of EDAC, i.e.,1-ethyl-3(3-dimethylaminopropyl)carbodiimide), is added to the mixtureand the latter is allowed to incubate overnight. The pH of the mixtureis maintained between 5.5 and 6.

The incubated material is then dialyzed against distilled water for 3days. This material is then mixed with Freund's complete adjuvant andinjected at multiple sites on the dorsal lateral surface of four goats.Two of the goats developed the appropriate antibody, which was recoveredby conventional means.

In plate preparative procedures, a commercially available Cookemicrotiter plate is first washed with deionized water and air dried. Theplate wells are then coated with a site-deactivating medium, in thiscase goat plasma. Whole goat plasma is first diluted 1:20 (V:V) with a0.1 Molar phosphate buffered saline (PBS) solution (pH7) containing 0.05Molar urea. Five milligrams of EDAC per milliliter is next added to thediluted goat plasma, and 200 μl of the final mixture is added to eachplate well. The plate is then incubated overnight at room temperature,whereupon it is decanted and washed six times with deionized water.

The propionic acid analogue of thiazide,3-(β-carboxyethyl)-6-chloro-7-sulfamoyl-1,2,4-benzothiadiazine-1,1-dioxide,(the antigen of the antigen-antibody reaction) is then covalentlycoupled to the goat plasma coating. This is accomplished by adding toeach plate well 200 μl of a solution of 5 mg. of EDAC and 0.25 mg of thethiazide analog per milliliter of the phosphate buffered salinesolutions. The plate is then incubated overnight at room temperature.Following incubation, the plate is decanted and washed six times withdeionized water. The plate may be stored wet or dry.

In testing procedures a reaction mixture comprising a sample fraction of25 μl of test urine and two hundred μl of the horseradish peroxidase(HRP) labeled anti-thiazide antibody in PBS is added to each well. Themixture is then agitated to insure proper mixing, and allowed to reactat room temperature for about 20 minutes.

The respective reaction mixtures are then dumped, and the plate iswashed six times with deionized water and shaken dry. Three hundredmicroliters of a known color-generating substance, 0.018 Molar ABTS(2,2'-azino-di-(3-ethyl-benzythiazoline sulfonic acid) diammonium salt)and 1.0 micromolar H₂ O₂ in 0.1 M phosphate (pH 6), is added to eachplate well. The plate is then incubated at room temperature and is readby visual observation for the presence or lack of color. Positives(thiazides present) are colorless to light green, whereas negatives (nothiazides present) will be dark green in color.

Having thus described the invention, what is claimed as new and desiredto be secured by Letters Patent is:
 1. In an assay process for thedetermination of an immunochemical component in a sample, wherein thesample, a first immunochemical component and a second immunochemicalcomponent are immunochemically incubated in a vessel, one of said firstand second components being insolubilized in said vessel, one of saidfirst and second components being labeled, said first and secondcomponents each having a specific immunochemical affinity for anothercomponent in said vessel, or for said component to be determined, duringsaid incubation thereof, the insolubilized component is separated fromthe remainder of the reaction mixture, and it is thereafter determinedwhether said sample contained said component to be determined, theimprovement which comprises:covalently bonding to the walls of saidvessel a site-deactivating medium for minimizing nonspecificinteractions, said insolubilized component being covalently attached tosaid medium, and said medium being selected from the group consisting ofwater soluble animal-derived plasma, animal-derived sera,vegetable-derived gelatins and mixtures thereof.
 2. The process as setforth in claim 1, wherein said immunochemical component to be determinedis an antigen.
 3. The process as set forth in claim 1, wherein saidlabeled immunochemical component is an antibody.
 4. The process as setforth in claim 1, wherein the label of said immunochemical component isan enzyme.
 5. The process as set forth in claim 1, said enzyme beinghorseradish peroxidase.
 6. The process as set forth in claim 1, saidcomponent-determining step comprising determining the activity of thelabeled component.
 7. The process as set forth in claim 1, saiddetermination being performed colorimetrically.
 8. The process as setforth in claim 1, said component to be determined being a thiazideantigen.
 9. The process as set forth in claim 1, wherein said firstcomponent is covalently coupled to said medium, said sample and labeledimmunochemical component being thereafter simultaneously placed in saidvessel.
 10. The process as set forth in claim 1, said firstimmunochemical component being an analogue of said component to bedetermined.
 11. A system for determining the presence of animmunochemical component taken from the group consisting of antigens andantibodies in a sample, comprising:a vessel having the internal wallsthereof covalently bound with a site-deactivating medium, said mediumbeing selected from the group consisting of water soluble animal-derivedplasma, animal-derived sera, vegetable-derived gelatins and mixturesthereof; an amount of a first immunochemical component covalently boundto said medium; and an amount of a second unbound immunochemicalcomponent, one of said first and second components being labeled, saidfirst and second components being present in respective amountssufficient to perform the assay process of claim
 1. 12. The system asset forth in claim 11, said first component being an analogue of saidcomponent to be determined.
 13. The system as set forth in claim 11,said label being an enzyme.
 14. The system as set forth in claim 13,said enzyme being horseradish peroxidase.
 15. The system as set forth inclaim 13, including a color-generating agent reactive with said enzymeto generate an analytically measurable signal.
 16. The system as setforth in claim 15, said antibody having an immunochemical affinity forsaid component to be determined.
 17. The system as set forth in claim11, said labeled component being an antibody.
 18. The system as setforth in claim 11, said first component being directly covalently boundto said medium.